抗酸染色阳性痰涂片刮取物扩增分枝杆菌基因的初步探索

发布时间：2019-07-01 12:15

【摘要】：背景 结核病是危害全球健康的重大公共卫生问题，虽随着短程督导化疗方案的普遍实施，结核病疫情已经有所控制，但当前结核病控制仍面临着耐多药，合并HIV感染及流动人口等问题带来的巨大挑战。非结核分枝杆菌是指结核与麻风分枝杆菌以外的其他分枝杆菌，是一类在自然界中广为分布的环境病原体。其可通过多种途径感染易感人群引起局部或全身病灶，甚至导致死亡。国内外越来越多的研究表明非结核分枝杆菌感染及其相关疾病的流行已呈现出上升的趋势。 抗酸染色涂片镜检法简单、快速，且是发现肺结核传染源的有效方法。在结核病高发地区，其已成为结核病实验室诊断的主要指标。但其只能证明抗酸杆菌的存在，不能区分结核与非结核分枝杆菌，在全球非结核分枝杆菌感染上升的背景下这使越来越多的涂阳病人有可能被误诊而接受不必要或不适当的抗结核治疗。依靠培养的传统菌种鉴定方法虽能达到区分两者的目的，但在发展中国家的多数基层实验室尚无技术条件开展培养实验，上级分枝杆菌实验室虽能开展，但其繁琐又费时，远不能满足临床诊疗的需要。 目的 了解NTM的流行状况和流行病学特征，初步评价IS6110荧光定量PCR法快速鉴定阳性痰涂片中的结核分枝杆菌的临床应用价值，同时对抗酸染色阳性痰涂片刮削物扩增分枝杆菌利福平耐药基因（rpoB基因）进行初步探索。 第一部分广州市越秀、海珠区非结核分枝杆菌流行状况分析 一、研究对象及方法 选取2010年-2012年于越秀、海珠区两个结核病防治所就诊的可疑肺结核患者，收集其痰分枝杆菌培养等实验室资料。对于在研究期间有多次阳性培养和菌菌种鉴定的同一病例只按1例计算，且选择在时间上靠近研究起点的实验室结果纳入统计分析。 每个病例均在清晨留取深部痰标本进行分枝杆菌液体培养检测，培养分离的菌株通过PNB生长抑制试验进行MTC与NTM初步鉴别，NTM分离株按照中国防痨协会操作规程通过分枝杆菌的培养特征及一系列生化测试鉴定其菌种。 二、结果 1、可疑肺结核患者痰标本中MTC与NTM的分离率分别为80.9%、19.1%。2010-2012各年度的NTM分离率分别为17.6%、17.1%、21.2%。 2、NTM分离者的男女比例为1.56：1，60岁及以上老年病例仅占26%，45-59岁、15-29岁年龄段为NTM分离高峰。 3、NTM菌种中快生长型占50.6%，慢生长型占49.4%。龟脓肿和鸟胞内复合群为主要的分离菌种，分别占40.5%和24.1%。 第二部分痰涂片抗酸染色阳性患者分枝杆菌的分离情况分析 一、研究对象及方法 选取2012年~2013年于越秀、海珠区两个结核病防治所诊治的痰AFB涂片阳性且同期培养阳性的病例共331例。其中男性238例，女性93例(M/F=2.56)；年龄范围15~87岁之间，平均39岁；初治225例，，复治106例。 所有病例均于清晨留取深部痰标本用于涂片抗酸染色及分枝杆菌分离培养和鉴定。 二、结果 1、痰涂片AFB阳性患者中MTC和NTM的分离率分别为81%和19%。2012年NTM分离率为14.1%，2013年为25.2%。 2、初治和复治涂阳患者NTM分离率分别为12%和34%。 3、MTC组涂片阳性等级≤1+占55.97%，1+占44.03%。NTM组涂片阳性等级≤1+占82.54%，1+占17.46%。 第三部分IS6110-荧光定量PCR法快速鉴定抗酸染色阳性涂片中的结核分枝杆菌 一、研究对象及方法 331例同期培养阳性的抗酸染色阳性痰涂片来自2012年-2013年于越秀、海珠区结核病防治所就诊的患者。其中阳性等级为1-9条/300视野的涂片25例，1+的涂片177例，2+涂片66例，3+涂片30例，4+涂片33例。根据其同期培养分群鉴定结果分为两组，其中MTC组268例，NTM组63例。 应用结核分枝杆菌(TB)核酸检测试剂对所有痰涂片刮削物分枝杆菌DNA行荧光定量PCR检测。 二、结果 1、荧光定量PCR法初步鉴定阳性涂片中分枝杆菌菌群的灵敏度和特异度分别为69.40%、76.19%。 2、荧光定量PCR法在鉴定抗酸染色涂片阳性标本中的分枝杆菌菌群与培养法一致程度较差（kappa=0.324）。 3、MTC组各阳性等级涂片的阳性检出率分别为26.67%、63.70%、68.97%、92.86%、93.75%。 4、FQ-PCR检测的靶基因拷贝数和涂片阳性等级存在正相关（r=0.563）。第四部分抗酸染色阳性痰涂片刮削物扩增分枝杆菌rpoB基因的初步探索一、研究对象及方法 18株临床分离结核分枝杆菌来自广州市胸科医院“广州地区分枝杆菌菌株库”，80张抗酸染色阳性(3+～4+)痰涂片来自广州市越秀区结核病防治所，涂片样本来自经分枝杆菌培养鉴定证实的肺结核患者。 设计两对引物，以菌株、痰涂片刮削物中提取的基因组DNA为模板扩增rpoB基因，PCR产物经凝胶电泳检测。 二、结果 1、两对引物对18例临床分离株基因组DNA进行扩增，均能见到明亮目的条带，扩增产物经测序证实为rpoB基因。 2、痰涂片刮削物DNA经不同模板量、不同退火温度，两组引物分别行普通PCR、Touchdown PCR、巢式PCR、二次PCR扩增rpoB基因均未有目的条带。荧光定量PCR检测两种不同方法提取的痰涂片DNA拷贝数分别为102-103copies/ul和104-105copies/ul。 3、菌株基因组DNA经100-107一系列倍数稀释后，两对引物扩增rpoB基因分别在104、103以下的稀释倍数能观察到明亮目的条带，对应的荧光定量PCR检测拷贝数分别为106copies/ul、107copies/ul。 结论 1、可疑肺结核患者痰标本中NTM分离率19.1%。 2、NTM感染人群以中青年为主。 3、NTM以快速生长分枝杆菌较多见。 4、涂阳患者NTM分离率为19%,2013年涂阳患者NTM分离率明显高于2012年。 5、复治涂阳病人比初治者更易分离到NTM。 6、非结核分枝杆菌痰涂片阳性标本以1+以下多见。 7、IS6110-荧光定量PCR法检测到的结核分枝杆菌基因拷贝数与涂片阳性等级成正相关。 8、IS6110-荧光定量PCR法可作为阳性等级3+以上痰涂片中分枝杆菌快速初步鉴定的较好方法。 9、阳性痰涂片刮取物中提取的DNA量不足是rpoB基因未能成功扩增的主要原因。
[Abstract]:background Tuberculosis is a major public health problem that is harmful to the global health, although with the general implementation of the short-range supervision and chemotherapy scheme, the tuberculosis epidemic has been controlled, but the current tuberculosis control is still facing the problems of multi-drug resistance, the combination of HIV infection and the floating population. The non-tuberculous mycobacteria refer to the other mycobacteria other than the Mycobacterium tuberculosis and the M. jatropha. It is a kind of environmental pathogen that is widely distributed in nature. a body that can infect a susceptible population by a variety of ways to cause local or systemic lesions, or even death The more and more studies at home and abroad have shown that the prevalence of non-tuberculous mycobacteria infection and its related diseases has been increasing. The anti-acid staining smear microscopy method is simple and rapid, and is the source of the infection of the pulmonary tuberculosis. Effective method. It has become the principal of the tuberculosis laboratory diagnosis in a high-incidence area of tuberculosis. However, it can only prove that the presence of antacid can not distinguish between the tuberculosis and the non-tuberculous mycobacteria, and in the background of the rise of the infection of the non-mycobacterium tuberculosis in the world, the more and more smear-positive patients are likely to be misdiagnosed with unnecessary or inappropriate antiknot Nuclear therapy. Although the traditional identification method of the traditional culture can achieve the purpose of distinguishing the two, in most basic-level laboratories in developing countries, there are no technical conditions to carry out the culture experiment, but the superior mycobacteria laboratory can be carried out, but it is tedious and time-consuming and can not meet the clinical diagnosis and treatment. to be required Objective To study the prevalence and epidemiological characteristics of NTM, and to evaluate the rapid identification of Mycobacterium tuberculosis in the positive sputum smear by using the IS6110 fluorescence quantitative PCR method. Clinical application value of mycobacterium tuberculosis rifampin-resistant gene (rpoB gene) was amplified by anti-acid staining positive sputum smear. Preliminary exploration. The first part of Guangzhou Yuexiu, non-tuberculosis in Haizhu District M. M. prevalence I. The study object and method selected the suspected pulmonary tuberculosis patients treated by the two tuberculosis prevention and control centers in Yuexiu and Haizhu District in 2010-2012, and collected it. For laboratory data, such as the culture of Mycobacterium tuberculosis, only one case was calculated for the same case with multiple positive culture and bacterial strain identification during the study, and the selection was close to the study in time The laboratory results of the point were included in the statistical analysis. In each case, the deep sputum samples were collected in the morning for detection of mycobacteria liquid culture, and the isolated strains were inhibited by the growth of the PNB The MTC and NTM were initially identified by the test, and the NTM isolates were cultured in accordance with the operation procedures of the Chinese Anti-Japanese Association through the culture of Mycobacteria. features and one The isolation rates of MTC and NTM in sputum specimens of patients with suspected pulmonary tuberculosis were 80.9% and 19.1%, respectively. The ratio of the male to female was 1.56:1, the age of 60 and older was only 26%, and 45- 59-year-old,15-29-year-old was the peak of NTM separation. The fast-growing type (50.6%) and the slow-growing type (49.4%) in the species (49.4%). The main separation strains were 40.5% and 24.1%, respectively. The second part of the sputum smear is acid-fast Analysis of the isolation of M. positive patients with positive staining. The study object and method were selected from 2012 to 2013 in Yuexiu and Haizhu District. There were 331 cases of sputum AFB smear positive and positive in the same period, of which 238 cases were male and 93 cases were female (M/ F = 2.56). Between the ages of 15 and 87, the average age was 39; in the first treatment,225 cases were treated and 106 cases were retreated. All cases All in the morning The separation rate of MTC and NTM in sputum smear AFB positive patients was 81. % and 19%. The NTM separation rate in 2012 was 14.1%,2013 The rate of NTM was 12% and 34%, respectively. The positive grade of the smear positive of the MTC group was 55.97%. + accounted for 44.03%. The positive grade of smear positive in NTM group was 82.54%, and 1 + was 17.46%. . Part III IS Rapid identification of Mycobacterium tuberculosis one, study object and method in acid-fast stain positive smear by 6110-fluorescence quantitative PCR The positive anti-acid stained positive sputum smear from 2012 to 2013 in Yuexiu, Haizhu District, for the prevention and treatment of tuberculosis, in which the positive grade is 1-9/ There were 25 smear of 300 eyes,177 cases of 1 + smear,66 cases of 2 + smear and 30 cases of 3 + smear. And 33 cases of 4 + smears were divided into two groups according to the group identification results of the same period, including 268 cases of MTC group and 63 cases of NTM group. .. Detection of the fluorescence quantitative PCR of Mycobacterium tuberculosis DNA by using Mycobacterium tuberculosis (TB) nucleic acid detection reagent. 1. The sensitivity and specificity of the bacterial flora in the positive smear were 69.40% and 76.19%, respectively. The fluorescence quantitative PCR method has a poor degree of consistency with the culture method (kappa = 0.324) in the identification of the positive specimen of the acid-fast stain smear. The positive rate of positive grade smear in MTC group was 26.67%, 63.70% and 68.9 respectively. 7%, 92.86%, 93.75%.4, FQ-PCR-detected target gene copy Number and smear positive, etc. There was a positive correlation (r = 0.563) in the stage. The fourth part of the acid-fast staining positive sputum smear was used to amplify the rpoB gene of the Mycobacterium tuberculosis. >,80 anti-acid staining positive (3 + ~ 4 +) sputum smear from the tuberculosis prevention and control department in Yuexiu District, Guangzhou, and the smear samples from the culture and identification of the mycobacterium tuberculosis solid lung junction designing two pairs of primers to amplify the rpoB gene by using the genomic DNA extracted from the bacterial strain and the sputum smear scraper as a template, 2. Results 1 and 2 pairs of primers were used to amplify the genomic DNA of 18 clinical isolates. A. The rpoB gene was amplified by ordinary PCR, Touchdown PCR, nested PCR and secondary PCR with different template and different annealing temperatures. the DNA copy number of the sputum smear extracted by the two different methods is 102-103 copies/ ul and 104-105 copies/ ul.3, and the two pairs of primers amplify the rpoB gene respectively after the bacterial strain genomic DNA is diluted by a series of multiple times of 100-107, at 1 the dilution factor below 04,103 can be observed for a bright target strip, for example, The copy number of the fluorescence quantitative PCR should be 涓

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